RNA polymerases are nucleotidyl transferase enzymes that produce RNA by polymerizing ribonucleotides at the 3′ end of an RNA transcript. In cells, RNA polymerases are needed for constructing RNA chains from DNA or RNA templates, a process called transcription. Control of the process of transcription affects patterns of gene expression and thereby allows a cell to adapt to a changing environment, perform specialized roles within an organism, and maintain basic metabolic processes necessary for survival. Therefore, it is hardly surprising that the activity of RNA polymerases is both complex and highly regulated. Examples of products of RNA polymerases include messenger RNA (mRNA), non-coding RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), microRNA (miRNA), and catalytic RNAs such as ribozymes.
RNA polymerases are essential to life and are found in all organisms and many viruses. Of the RNA polymerases in eukaryotes, RNA Polymerase I (also called Pol I) is the enzyme that transcribes ribosomal RNA (excluding 5S rRNA, which is synthesized by RNA Polymerase III), which accounts for over 50% of the total RNA synthesized in a cell. RNA polymerase II (also called RNAP II and Pol II) catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most-studied type of RNA polymerase. A wide range of transcription factors are required for RNAP II to bind to its promoters and begin transcription. RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of “housekeeping” genes whose expression is required in all cell types and most environmental conditions. Thus, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, requiring fewer regulatory proteins than RNA polymerase II.
RNA polymerases can initiate transcription at specific DNA sequences known as promoters, producing an RNA chain complementary to the template DNA strand. The process of adding nucleotides to the RNA strand is known as elongation. In eukaryotes, RNA polymerases can build chains millions of nucleotides in length. RNA polymerases will preferentially release the RNA transcript at specific DNA sequences encoded at the end of genes known as terminators.
Many viruses also encode RNA polymerases. Perhaps the most widely studied viral RNA polymerase is found in bacteriophage T7. The single-subunit T7 RNA polymerase is related to that found in mitochondria and chloroplasts, and shares considerable homology to DNA polymerase. It is believed that most viral polymerases therefore evolved from DNA polymerase and are not directly related to the multi-subunit polymerases described above.
Viral polymerases are diverse, and include some forms which can use RNA as a template instead of DNA. This occurs in negative strand RNA viruses and dsRNA viruses, both of which exist for a portion of their life cycle as double-stranded RNA. However, some positive strand RNA viruses, such as polio, also contain these RNA-dependent RNA polymerases.
RNA-directed transcription refers to transcription from an RNA template and is carried out by an RNA-dependent RNA polymerase (RdRP). RNA-directed transcription occurs in RNAi-mediated gene silencing in non-vertebrate organisms such as C. elegans, fungi, and plants (Wassenegger, M. and Krczal, G. Trends Plant Sci. 11:142-51 (2006)). RNA-directed transcription is also part of the normal replication cycle of a number of vertebrate RNA viruses. All these examples of RNA-directed transcription involve a dedicated RdRP. In the case of virus replication, the RdRP is virally encoded.
Hepatitis Delta Virus (HDV) is the smallest known animal virus and encodes only one protein, the Hepatitis Delta Antigen (HDAg). HDAg is required for viral replication, but does not have polymerase activity. HDV is naturally acquired either by co-infection with Hepatitis B Virus (HBV) or by super-infection of a host with an existing HBV infection. The role of HBV in the natural HDV life-cycle relates to the Hepatitis B surface antigen (HBsAg) which forms part of the HDV envelope and is responsible for hepatocyte-specific infection. However, once inside essentially any mammalian cell, HDV replication is independent of HBV, so long as HDV RNA and a source of HDAg are provided.
Following infection, the HDV RNA-HDAg ribonucleoprotein particle (RNP) is recruited to the nucleus where the circular genomic HDV RNA serves as a template for rolling-circle replication, thereby generating multimers of antigenomic HDV RNAs. As HDV does not encode a protein with polymerase activity, virus genome replication relies on host RNA polymerases (Taylor, J. M. Curr. Top. Microbiol. Immunol. 307:1-23 (2006)). The multimers of antigenomic HDV RNAs are cleaved into monomers by a ribozyme activity in the antigenomic RNA, which then circularises by end-ligation. The resulting antigenomic monomers then become templates for analogous rolling-circle transcription, thereby yielding more circular genomic HDV RNAs. Due to the presence of >70% intramolecular Watson-Crick base-pair complementarity, both genomic and antigenomic HDV RNA assume a compact, unbranched rod-like structure.
In addition to being the template for antigenomic RNA synthesis, genomic HDV RNA also serves as the template for transcription of HDAg mRNA. This mRNA is capped and polyadenylated as is typical for RNA Polymerase II (Pol II) transcripts (Hsieh, S. Y. et al. J. Virol. 64:3192-8 (1990); Gudima, S. et al. J. Virol. 74:7204-10 (2000). For this reason, and the additional observation that both genomic and antigenomic HDV RNA can be immunoprecipitated with Pol II antibodies (Greco-Stewart, V. S. et al. Virology 357:68-78 (2007)), it appears that the Pol II core enzyme mediates HDV RNA-directed transcription. However, the mechanism behind HDV replication and transcription remains poorly understood.